The eukaryotic ATP-ases of SMC family (structural maintenance of chromosomes) form several essential eukaryotic protein complexes that determine the higher-order chromosome structure and dynamics in eukaryotic cells. One of these complexes, termed condensin, is in the current focus of studies by the Unit of Chromosome Structure and Function. Condensin complex constitutes the main molecular machinery of chromosome condensation, a process indispensable for proper separation of sister chromatids and their segregation during anaphase. In budding yeast and higher eukaryotes condensin is composed of five essential subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4 and Brn1. At present, the molecular mechanism of condensin activity in vivo is unknown. In order to understand the essence of condensin activity in chromatin the studies in the Unit were focused on the particular aspect of this activity: the specificity of condensin targeting to the natural chromatin sites in budding yeast. Previous studies in the Unit identified nucleolar chromatin (the rDNA genomic locus) as the major binding site for condensin in S. cerevisiae. In addition, existence of non-nucleolar binding sites was suspected, based on genetic and cell biology data. The role of genetic and epigenetic factors in recognition of specific chromosomal domains by condensin was investigated. With this general goal three specific research directions were followed: (i) investigation of the genomic distribution and cell-cycle control of condensin binding outside of the nucleolus, (ii) screening for molecular mechanisms determining specificity of mitotic condensin targeting to nucleolar chromatin, (iii) analysis of posttranslational modifications in condensin binding regulation. (i) Whole-genome analysis of condensin-binding sites was conducted to identify condensin-bound DNA fragments: immunoprecipitation of chromatin-bound condensin was followed by hybridization to genomic microarrays (ChIP-chip analysis). It was established that condensin is distributed over chromosomal arms, with strong binding peaks approximately every 10 kb. Thus, numerous novel sites of condensin binding were identified. Quantitative PCR validation confirmed that condensin occupies sites across the genome and in the specialized chromatin regions: near telomeres, in heterochromatic regions and in the zones of converging DNA replication. Comparison of condensin binding throughout the cell cycle revealed a cyclical mode of condensin binding at some sites. In mitosis condensin was enriched at rDNA, subtelomeric and pericentromeric regions. (ii) The genetic studies in the Unit established that several pathways determine proper condensin localization to the specific chromatin domains and thus facilitate/regulate the chromosome condensation process. Screening for molecular mechanisms determining specificity of mitotic condensin targeting to nucleolar chromatin was focused on elucidating the role played in condensin affinity by the repeating nature of the rDNA locus. Four condensin binding sites within the 9-kb rDNA unit were found to be controlled by two independent pathways, independently involved in the complex process of maintaining the nucleolar organizer. The molecular components of these pathways are under investigation. (iii) Analysis of posttranslational modifications in regulation of condensin binding to chromatin, particularly to pericentromeric regions, was focused on the SUMO(Smt3)-modification machinery. Functional interaction between sumoation machinery and condensin was previously discovered in the Unit. This pathway mediates chromosome condensation and condensin targeting to chromatin. While it was demonstrated that condensin itself is not modified by Smt3 when bound to chromatin, topoisomerase II, which interacts with condensin both physically and functionally, can be efficiently modified by Smt3. The studies in the Unit established that Smt3 modifications at the Top2 tail can alter targeting of this enzyme within the cell, in a chromatin-domain specific manner, dependent on the number of Smt3 molecules attached. Moreover, genetic analysis of interaction between Top2 modification and the genes encoding the Smt3 E3 (SIZ1 and SIZ2) demonstrated that the role of these genes in chromosome stability is mediated by Top2. Genetic interaction was also found between these two genes and condensin mutations. The molecular interface between sumoylated Top2 and condensin is being investigated.